1. Field of the Invention
The present invention discloses a method and composition for detecting the presence of antibodies in human or animal bodily fluids (blood, serum, plasma, urine, colostrum, milk, tears, or saliva) to analytes such as bacteria, Chlamydiae, Rickettsiae, protozoa, allergens, autoimmune antigens, viral proteins, and carbohydrates by lateral flow techniques.
2. Description of the Prior Art
Over the years, numerous patents have been issued involving immuno-chromatographic devices. The standard features of these devices comprise the following:
a) A plastic or paper housing allowing the viewing of a reaction area on a bibulous (lateral flow) strip;
b) An opening at one end of the housing allowing for the addition of sample (urine, blood, plasma, serum or bacteria in a media base);
c) Bibulous material (the lateral flow strip) having immobilized specific binding members (analytes) capable of reacting with antigens or antibodies.
d) A pad of absorbent bibulous material (the absorbent pad) enclosed at the end opposite the sample well and used to absorb transversely flowing sample, buffers and colloids;
e) A strip of bibulous material used in the sample well end to initially absorb the sample being applied;
f) A strip of bibulous material in contact with the sample well material and the lateral flow strip and containing a dried colored solid phase reagent, the solid phase coated with proteins or haptens.
Two types of chromatographic immunoassays are commonly described. In the one, proteins or small molecule analytes contained in human fluids (urine, blood, plasma, serum, and saliva) are detected. The analytes include hCG, FSH, LH, CKMB, TSH, troponins, myoglobulin, cancer proteins, viralbacterial proteins, haptens, therapeutic drugs, and drugs of abuse.
In the other chromatographic immunoassay, the analyte being detected is human antibody specifically reactive with agents such as viral/bacterial proteins (HIV, Hepatitis A and C, H. pylori, EBV, Rubella, CMV, HSV, Dengue fever, Lyme, Chagas, TB, Toxoplasma, autoimmune antigens, etc.) or allergens (pollens, molds, dust/mites, foods, animal epithelia, etc.). The various analytes are abbreviated VB for simplified use below. When it comes to detecting antibody, three formats are typically used:
1) The colored solid phase [SP] is coated with proteins or lectins [protein A, protein G, lentil lectin, jacalin, concanavilin A, mannan binding protein, wheat germ lectin, peanut lectin and avidchrom] that react with human IgG antibodies. The solid phase may be coated with anti-immunoglobulins that specifically react with IgG, IgM, IgA, or IgE. The bibulous strip would in this case contain the analyte (VB) of interest to which the specific antibody contained in the sample reacts.
2) The colored solid phase contains the analyte (VB) to which the human immunoglobulins react. The bibulous strip would in this case also contain the analyte (VB) of interest to which the specific antibody contained in the sample reacts.
3) The colored solid phase contains the analyte (VB) to which immunoglobulins react. The bibulous strip contains proteins directed against human IgG or total immunoglobulins (protein A, protein G, lectins, lentil lectin, jacalin, concanavilin A, mannan binding protein, wheat germ lectin, peanut lectin and avidchrom or a mix of antibody to immunoglobulin classes IgG, IgA, IgM and IgE).
U.S. Pat. No. 5,459,041 (Blaser et al.) discloses antigenic compositions for use in diagnostic kits and the like for detecting the presence of antibodies specific for Campylobacter pylori, Samples of bodily fluids, for instance, may be contacted with immobilized antigen on a solid phase which is then washed and tested for the occurrence of significant levels of antigen/antibody complex. Levels exceeding a predetermined positive threshold are indicative of antibodies to Campylobacter pylori in the sample tested. Kits employing the antigenic compositions of the invention preferably include means for detecting the antigen/antibody complex such as materials and reagents for conducting an enzyme-linked immunosorbent assay, Western blot technique, ELISA, liposome-based assay or other known detection tests. The Western blot and ELISA tests used here are for the detection of IgA and IgG antibodies.
U.S. Pat. No. 5,567,594 (Calenoff) discloses a library of isolated and purified antigens specific for a microorganism is a set of individual molecules. The library forms antigen-antibody complexes useful in the context of diagnosing and treating conditions associated with a specific microorganism such as H. pylori-induced gastro-duodenal disease. For the antigen-antibody complexes the antibody in question is an immunoglobulin, which is IgE if the antigens are allergens. Antigen-antibody complexes with IgA, IgG and IgM are also useful if the antigen is a bacteria. By this multivariate approach, a specific condition is diagnosed with high sensitivity and specificity by determining whether complexes form between a specific antigen library and a biological sample which contains immunoglobulins from an individual. Such libraries also are useful for immunotherapy. Western blot is used to detect IgE antibodies. The method requires enzyme conjugates and enzyme substrates and two wash steps to detect antibodies.
U.S. Pat. No. 5,420,014 (Cripps et al.) discloses a method for detecting a current infection by H. pylori in a mammal. The method comprises contacting a mucous secretion [saliva] from said mammal with an immobilized antigen component from H. pylori for a time and under conditions sufficient for an IgG antibody in said mucous secretion specific to a antigen component to form a complex therewith and then subjecting said complex to a detecting means which involves an enzyme conjugate and specific substrate.
U.S. Pat. No. 6,068,985 (Cripps) discloses a method which uses saliva to detect IgG in both the Western Blot and ELISA tests. This detection method requires the use of an enzyme conjugate and enzyme substrate and two wash steps to detect the antibody.
U.S. Pat. No. 5,846,751 (Pronovost et al.) discloses a sensitive and specific antigen preparation for the detection of Helicobacter pylori in biological samples. The preparation uses a range of antigens derived from size exclusion chromatography of detergent-solubilized H. pylori cells and the purified antigen preparation is coated on the solid phase. Serological assays such as ELISA, latex agglutination, and rapid EIA assays are used to detect antibodies to H. pylori. The invention also uses a lateral flow device to detect total immunoglobulins to H. pylori. In this case, the H. Pylori antigen is striped on the membrane reaction area and also coated to the colored solid phase. The antibody in the sample reacts first with H. pylori gold coated conjugate, and then travels to the membrane reaction area where it reacts with striped H. pylori. 
U.S. Pat. No. 5,200,344 (Blaser et al) uses a purified p28kd protein from H. pylori to detect IgA, IgM and IgG antibody in ELISA and Western Blot. The test requires conjugate and enzyme substrate and two wash steps to detect the antibody.
U.S. Pat. Nos. 6,060,326 and 5,945,294 (Frank et al.) discloses methods to detect canine IgE using a canine Fc epsilon receptor to detect canine IgE antibodies in a biological sample from a canine.
U.S. Pat. No. 5,547,833 (Dorval et al.) discloses a radial flow assay delivery device, and methods of use.
None of these patents teach or disclose a fast and effective lateral flow assay test for the testing of multiple-class specific antibodies. More specifically, no chromatographic immunoassay is able to distinguish between reactive antibody contained in the classes of human antibody (IgG, IgA, IgM, IgD and IgE). All devices to date detect either total immunoglobulins or IgG. The problem of separating reactivities of antibody class lies in the 10 to 15 fold excess of IgG class specific antibodies over IgA, IgM, and IgE class specific antibodies reactive with analyte (VB) in question at various protein sites (epitopes). If the IgG is allowed to react at the same time or same rate as other classes of antibody, the IgG will mask most if not all the analyte (VB) epitopes, thereby decreasing or eliminating the activity of the IgM, IgA, and IgE class antibodies to the analyte (VB).
The proposed invention allows for antibody class recognition. In one embodiment of the invention, a lateral flow immunoassay device distinguishes at least three classes of antibody. The classes of antibody to be distinguished include IgG, IgA and IgM. A control line reactive with gold particles is also present.
In another embodiment of the invention, the immunoassay test strip is modified to allow detection of the IgE class of antibody to many allergens (VB) coated sequentially on a bibulous strip. Saturated anti-IgE antibodies coated to colored solid [SP] phase particles at high concentration are reacted with a controlled amount of serum to allow for the near complete complexing of elevated levels of human or other animal IgE. This insures that little free IgE is left unreacted. Unreacted IgE would inhibit the reaction with the multiple analytes coated on the bibulous strip. By capturing most of the IgE on the colored solid phase, sufficient IgE specific antibody molecules are available to react with the various allergens (VB) as the reaction front moves transversely down the strip toward the absorbent pad. This allows for the detection of many different IgE allergen specific molecules.
In both embodiments of the inventions, an IgG reacting protein (which can be protein A, protein G, an antibody to IgG or lectins such as lentil lectin, jacalin, concanavilin A, mannan binding protein, wheat germ lectin, peanut lectin and avidchrom) is added to the sample pad in order to complex the IgG contained in the sample such that the molecular weight of the IgG complex is greater than 1.0 million. This large complex travels sufficiently slower than IgA, IgM, and IgE thereby allowing these antibodies to react prior to the IgG. After reacting to the antigen coated colored solid phase, the various reacted complexes are captured on the bibulous strip coated at three sites with antibody to IgM, IgA and IgG or a protein/lectin reactive with IgG (protein A, protein G, lentil lectin, jacalin, concanavilin A, mannan binding protein, wheat germ lectin, peanut lectin and avidchrom). Thus, the class of reactive antibody is distinguished.
In another embodiment of the invention, the colored solid phase contains proteins that react with IgG, allowing for the detection of many different analyte specific antibody molecules of the IgG class. The reagents coated onto the bibulous lateral flow strip include autoimmune antigens, allergens, Chlamidia, Rickettsiae, viruses, and bacteria.